Methylation patterns are now appreciated as an important component in the complex web of mechanisms which ensure the orderly unfolding of the developmental program, and it is clear that disturbances in the DNA- methylating system may disrupt normal patterns of cell growth and differentiation. The long-term goal of the proposed research is an understanding of the regulation of DNA methylation. Methylation patterns are established and maintained by DNA (cytosine-5)-methyltransferase (DNA MTase). In previous work this enzyme was purified to homogeneity from extracts of mouse nuclei, characterized in terms of sequence specificity and other properties, and the full-length cDNA which encodes it cloned and sequenced. The proposed research will use the cloned DNA MTase sequences to address questions regarding the role of DNA MTase in cellular differentiation, and to elucidate structure/function relationships in the enzyme itself. Specific lines of experimentation include the following. (1) The C-terminal 1/3 of the 1573-amino acid DNA main. This domain will be produced by expression of a truncated coding region in heterologous cells, and the isolated C-terminal domain compared to the intact enzyme in terms of de novo sequence specificity, relative preference for hemimethylated and unmethylated DNA, and specific activity (kcat). (2) The N-terminal 1000 amino acids of DNa MTase may represent a regulatory domain. A Cysteine-rich region similar to Zn-binding sites characteristic of many regulatory proteins is located in the N-terminal domain of DNA MTase; potential metal-binding residues will be eliminated by site-directed mutagenesis and the properties of mutant enzymes compared to those of the intact enzyme as described in (1) to test the function of the Cys-rich region. The function of a suspected specificity-determining region within the C-terminal domain will be examined by sequence exchanges with bacterial restriction methyltransferases. (3) Synthesis of DNA MTase will be selectively suppressed in vivo by means of complementary RNA; inhibition of methylation by this method is predicted to induce differentiation of murine erythroleukemia and C3H10T1/2 cells, which have been reported to have obligate demethylation events in their differentiation pathways. A sensitive new demethylation assay will be used in these studies. (4) Certain cell types are very active in de novo methylation and may contain undescribed species of DNA MTase; RNA from these cells will be searched for new species of DNA MTase by means of reverse transcription and polymerase chain reaction amplification in the presence of oligonucleotide primers corresponding to peptide sequences that are conserved among all DNA cytosine methyltransferases. (5) In collaboration with R. Jaenisch (Whitehead Institute and Department of Biology, MIT), the DNA MTase locus will be inactivated in mice via gene targeting in embryonic stem cells. This work will test the dispensability of DNA MTase in homozygous mutants, and any new phenotypes that arise in heterozygotes may help to identify processes in which methylation patterns play an important role. DNA MTase plays a central role in an important regulatory system, and the studies proposed here will illuminate important aspects of both the biological role and the function of this enzyme.